3D localization microscopy (such as PALM or STORM) is used to obtain three dimensional structure information of organic or inorganic targets such as molecules in an imaging area to determine the relative locations of the targets, and the resulting location information is used to create a single 3D image or representation, typically to an in plane resolution of better than 30 nanometers.
The imaging area is filled with many fluorescent markers, that are illuminated to fluoresce and the location of each determined. Using an appropriate illumination protocol only some markers emit fluorescence at a given time and are recorded in a single image frame. The location of those markers which fluoresce is determined, and the location information from multiple such images of various random marker sets is assembled into the single 3D image or representation. Different methods are known for obtaining not only x and y axis but also z axis location information from the 2D images.
The microscope resolution is limited by the optical diffraction limit of the microscope. Diffraction causes a single point emitter of a size less than the diffraction limit of the microscope to be imaged as a larger spot. Using algorithms such as fitting or centroid determination, and knowing the point spread function (PSF) of the microscope, it is possible to determine the centre of the spot to beyond the diffraction limit in the x and y axes.
Current 3D localization strategies generally fall into two categories: acquisition of multiple simultaneous observations of the molecule, as in the biplane and iPALM approaches, or methods that engineer the point spread function (PSF) such that 3D position information is encoded in the defocus dependent shape of the lateral PSF profile, as exemplified by the astigmatism and double-helix methods. In the double helix method the imaging system has a PSF engineered so that a single point emitter produces two separated image spots, in which the angle of a (notional) line between the two spots is dependent on the distance between the emitter and the objective. Changing the position of the emitter along the z axis of the objective causes the two image spots to rotate together i.e. the angle of the line between them changes, in a characteristic manner. Once calibrated, this rotational behaviour can be used to estimate the z-position of an emitter from a single 2D image without the need to move either the sample or the objective. U.S. Pat. No. 7,705,970 and US Patent Application Publication 2009/0219549 disclose such systems.